The hyperglycemic complications in diabetes and obesity are causally associated with overproduction of reactive oxygen species (ROS). A major source of ROS generation in hyperglycemic conditions is thought to be the mitochondrial electron transport chain. This proposal is to define the role of mitochondrial fission in regulating ROS production in hyperglycemic conditions. We have found that mitochondria undergo rapid fragmentation and recovery within an hour of exposure of cells to a high glucose concentration, which occurs concomitantly with an increase and decrease of ROS levels. In prolonged incubation in high glucose conditions, the ROS level increased again after the initial short burst and remained high for an extended period. We observed increased cell death through apoptosis after the prolonged incubation in high glucose, indicating that a continuous high level of ROS is the cause of hyperglycemic damage. Importantly, we found that blocking mitochondrial fragmentation by inhibiting mitochondrial fission prevents the high glucose-induced ROS overproduction. Although ROS-mediated damages in hyperglycemia have been studied extensively, how mitochondria overproduce ROS in hyperglycemic conditions is not well understood. Our findings indicate that mitochondrial dynamics are an important factor that regulates ROS generation in high glucose conditions. In this proposal, we will study how mitochondrial fission participates in the upstream and downstream processes of hyperglycemia-induced ROS production. Therefore, the Central Hypothesis of this proposal is that signals elicited from high glucose stimulation alter mitochondrial dynamics and structure, resulting in ROS production and cell injury. We will, first, investigate high glucose-mediated signaling pathways that regulate mitochondrial fission, second, study how altered mitochondrial morphology causes ROS overproduction in high glucose conditions, and third, test the proof-of-principle for mitochondrial fission as a potential target to reduce the hyperglycemia-induced oxidative damages. PUBLIC HEALTH RELEVANCE Information from these studies will expand the potential targets for controlling the hyperglycemia-induced damages in diabetes and obesity, and greatly increase our understanding of how mitochondrial dynamics contribute to the regulation of mitochondrial function and cell physiology in health and disease.